Fig 1.
Model of human embryonic stem cell directed differentiation into prostate tissue in vitro.
(A) Definitive endoderm and mesoderm differentiation was driven by culturing hESC with activin A (100 ng/ml) for 3 days. In the next 4 consecutive days, differentiated cells were cultured with WNT10B (500 ng/ml) and FGF10 (500 ng/ml) to direct them into prostatic fate and organoid formation. Organoids were transferred and grown in Matrigel to allow their 3-D growth in prostatic media containing T (1.7 μM) and ATRA (10 nM), which permits differentiation and expansion of prostatic organoids. (B) Definitive endoderm differentiation images show morphological changes at 24 and 72 hours following activin A treatment, compared to untreated hESC (control). Phase-contrast images were obtained using the EVOS microscope. Scale bars represent 200 μm. After 3 days of treatment with Activin A, Day 3 DE cultures were immunostained for (C) FOXA2 (red), an endodermal specific marker and (D) Brachyury (green), a mesendodermal marker with nuclear staining (DAPI, blue). The merged image (E) show definitive endoderm staining (red-yellow; arrowheads) in the majority of cells while a subpopulation was Brachyury+ but FOXA2- (arrows) implicating a mesodermal component in a minority of cells. Scale bars represent 50 μm.
Table 1.
Primary antibodies used for immunofluorescence.
Table 2.
Primer sequences used for real-time PCR.
Fig 2.
Directed differentiation into prostatic fate determination.
Prostatic fate determination images illustrate morphologic changes over 96 hours of treatment with 500 ng/ml WNT10B and FGF10 compared to untreated hESC as control shown in inset. After 48 and 72 hours of growth factor culture, spheroid-like structures attached to the cell monolayer were observed. At 96 hours, prior to Matrigel culture, 3-D structures with a budding-like phenotype were observed. Phase contrast images were obtained using the EVOS microscope. Scale bars represent 200 μm.
Fig 3.
Characterization of prostatic phenotype.
(A) Twenty-eight day time course phase-contrast images of a representative Matrigel cultured organoid forming over time from DE-differentiated H9 cells. While images of M-d1, d-4 and d-8 show the entire organoid as it grew, images of M-d20, d-24 and d-28 represent focal areas of formation and elongation of a single duct with extended culture. The representative duct is composed of a putative layer of columnar epithelium (arrows) with central lumens (dotted green lines), surrounded by mesenchyme (arrowheads). All images were obtained at the same magnification, scale bars represent 50 μm. (B) M-d16 and d-22 phase-contrast images following a representative Matrigel cultured organoid differentiated from H1 hESC. The organoid exhibited growth, budding, elongation and increased complexity over 6 days. Scale bars represent 50 μm. Insets: Lower magnification photographs show the entire organoid composed of convoluted ductal structures and mesenchyme (arrowheads). Scale bars represent 200 μm.
Fig 4.
Characterization of functional differentiation of organoids.
Immunostaining analysis by confocal microscopy of 28–30 day organoids for cytodifferentiation and functional differentiation markers. (A and D) Luminal cell cytodifferentiation markers AR (green) and (B and E) CK8/18 (red) and (C and F) merged images with DAPI (blue) reveals most luminal epithelial cells contain nuclear AR. (G) Merged NKX3.1 (green), a prostate specific epithelial cell marker, with CK8/18 (red), a luminal epithelial cell marker and DAPI (blue) shows nuclear NKX3.1 in all epithelial cells suggesting prostatic nature. Functional differentiation markers PSA (H and I) and androgen regulated gene TMPRSS2 (J) indicate the ability of cytodifferentiated luminal cells to produce secretory proteins specific to the prostate. Laminin (K), a basement membrane marker, delineates the normal acinar organization of the organoids. Staining for Vimentin (L) confirms the extra-acinar cells are derived from mesenchymally differentiated hESC cells, forming a stromal compartment. Normal IgG as negative controls for each probe is shown in insets. Lumens are indicated by white dotted lines (H-L) and epithelial ducts are outlined by green dotted lines (D-F). Scale bars represent 20 μm.
Fig 5.
BPA effects during prostatic organoid development.
Organoid quantitation (A-C) was performed 4 days following transfer to Matrigel in the absence or presence of 1 and 10 nM BPA. (A) 1 nM BPA increased budding (P < 0.001) whereas, 10 nM BPA reduced budding (P < 0.05) structure numbers compared to vehicle. (B) Nonbudding structure numbers were not affected by either dose of BPA. (C) While degenerating body numbers increased (P < 0.05) following 10 nM BPA exposure. (D) Differentiation gene expression of NKX3.1, CK18, AR and p63 was not altered by BPA treatment. In contrast, vimentin expression was significantly increased at 10 nM BPA (P < 0.01). (E) No difference in ERα, ERβ and GPER mRNA expression was noted. (F) Whereas, mRNA expression of 10 nM BPA treated organoids was significantly increased for the stem cell markers CD49f and NANOG (P < 0.05), with a similar trend noted for OCT4. TROP2 mRNA levels were not altered by BPA. From A-F bars represent means ± SEM (D-F n = 3), * P< 0.05, ** P < 0.01, *** P < 0.001. Scale bars represent 1 μm.
Fig 6.
BPA effects during prostatic organoid maturation.
Immunolocalization of the stem cell markers: (A) TROP2 and (B) CD49f show a dose-dependent increase in stem cell focal aggregates when treated with BPA compared to vehicle. (C) Organoids labeled with epithelial cytodifferentiation markers AR and CK8/18 show normal ductal morphology and epithelial differentiation after BPA treatment. Lumens are delineated by white and whole structures by green dotted lines. Scale bars represent 20 μm and all images are representative of n = 3.